Thermophilic L-carnosine hydrolase mutant and application thereof in preparation of L-carnosine

Abstract

The invention discloses a thermophilic L-carnosine hydrolase mutant and application of the thermophilic L-carnosine hydrolase mutant in preparation of L-carnosine, and belongs to the technical field of enzyme engineering and biology, the thermophilic L-carnosine hydrolase mutant is one of an N117E mutant, an I169L mutant or an N117E / I169L double mutant; the amino acid sequence of the N117E mutant is as shown in SEQ ID NO.17 in a sequence table, the amino acid sequence table of the I169L mutant is as shown in SEQ ID NO.19 in the sequence table, and the amino acid sequence table of the N117E / I169L double mutants is as shown in SEQ ID NO.21 in the sequence table. The carnosine hydrolase expressed by the thermophilic L-carnosine hydrolase mutant disclosed by the invention has excellent thermal stability and catalytic activity.

Description

Technical Field

[0001] The present invention relates to the fields of enzyme engineering and biotechnology, and specifically relates to a thermophilic L-carnosine hydrolase mutant and its application in the preparation of L-carnosine. Background Art

[0002] L-Carnosine is a natural dipeptide formed by the condensation of β-alanine and histidine. It not only has functions such as antioxidant and anti-inflammatory effects, but also has the activity of heavy metal chelation, so it has received extensive attention. Currently, the synthesis methods of carnosine mainly include chemical synthesis method and biosynthesis method. Among them, the chemical synthesis method requires the protection of the groups of the precursors β-alanine and histidine to avoid the generation of by-products, and then an activator is used to activate the carboxyl groups of the two amino acids to form an active intermediate. Finally, the two activated amino acids form a peptide bond reaction to synthesize L-carnosine. Due to its cumbersome reaction steps, many reaction condition limitations and high energy consumption, it will cause environmental pollution.

[0003] The biosynthesis method has the advantages of mild reaction conditions and no need for special conditions such as high temperature and high pressure, which can reduce environmental pollution. Among them, the enzymatic synthesis method has the advantages of high production yield and fast reaction speed, and has gradually received extensive attention. Currently, the enzymes that can synthesize carnosine mainly include aminopeptidase, carnosine synthase, L-amino acid ligase and carnosine hydrolase. Among them, carnosine synthase and L-amino acid ligase consume ATP during the synthesis of carnosine, which greatly increases the cost of synthesizing carnosine; aminopeptidase is an exopeptidase, which can reversely synthesize L-carnosine using β-alanine methyl ester and histidine as substrates; carnosine hydrolase can directly use β-alanine and histidine as substrates to reversely synthesize L-carnosine. This method does not require the modification of the precursors and does not require the participation of ATP during the reaction process, so it has received extensive attention.

[0004] Guan et al. disclosed in Molecular Catalysis in 2023 that through screening and molecular modification of proteins, a carnosine hydrolase SmPepD with high catalytic activity was obtained, and 31.3 mM of carnosine could be obtained after 8 h of reaction, but the optimal temperature of this enzyme was only 50 °C; Liu et al. disclosed in Bioresource Technology in 2023 that through the combination of carnosine hydrolase modification and transporter protein engineering, 29.1 g / L of L-carnosine could be produced after 48 h of reaction, but the optimal temperature of this reaction was only 45 °C.

[0005] The above results show that carnosine hydrolase has the ability to efficiently synthesize carnosine, but the current carnosine hydrolase has poor thermal stability. Therefore, there is an urgent need for carnosine hydrolase with good thermal stability and high catalytic activity. Summary of the Invention

[0006] To effectively solve the above problems, the present invention provides a thermophilic L-carnosine hydrolase mutant and its application in the preparation of L-carnosine. The L-carnosine hydrolase expressed by the thermophilic L-carnosine hydrolase mutant of the present invention has excellent thermal stability and catalytic activity.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions: A thermophilic L-carnosine hydrolase mutant, which is one of the N117E mutant, the I169L mutant or the N117E / I169L double mutant; The amino acid sequence of the N117E mutant is as shown in SEQ ID NO.17 in the sequence listing, the amino acid sequence of the I169L mutant is as shown in SEQ ID NO.19 in the sequence listing, and the amino acid sequence of the N117E / I169L double mutant is as shown in SEQ ID NO.21 in the sequence listing; Compared with the wild-type TcPepD enzyme, the asparagine at the 117th position in the amino acid sequence of the N117E mutant is mutated to glutamic acid; compared with the wild-type TcPepD enzyme, the isoleucine at the 169th position in the amino acid sequence of the I169L mutant is mutated to leucine; compared with the wild-type TcPepD enzyme, the asparagine at the 117th position in the amino acid sequence of the N117E / I169L double mutant is mutated to glutamic acid, and the isoleucine at the 169th position is mutated to leucine.

[0008] The nucleotide sequence of the N117E mutant is as shown in SEQ ID NO.18 in the sequence listing; the nucleotide sequence of the I169L mutant is as shown in SEQ ID NO.20 in the sequence listing; the nucleotide sequence of the N117E / I169L double mutant is as shown in SEQ ID NO.22 in the sequence listing.

[0009] The application of the thermophilic L-carnosine hydrolase mutant in the preparation of L-carnosine. After protein expression and protein purification of the thermophilic L-carnosine hydrolase mutant, a purified enzyme is obtained. Then, using β-alanine and L-histidine as substrates, a buffer solution with a pH of 6-10 as the reaction medium, and the purified enzyme is used for catalytic reaction at 50-80°C to obtain L-carnosine; The concentration of the β-alanine is 0.2-6M; The concentration of the L-histidine is 0.1-0.2M; The concentration of the purified enzyme is 0.04-0.06g / L; The rotation speed of the catalytic reaction is 1000rpm, and the reaction time is 1-24h; More preferably, MnCl2 was added to the catalytic reaction, and the concentration of MnCl2 was 1-100 mM.

[0010] Compared with the prior art, the present invention has the following beneficial effects: (1) The L-carnosine hydrolase mutant of the present invention is obtained by site-directed mutation on the basis of the wild-type Thermus thermophilus Thermobrachium celere After obtaining the mutant through site-directed mutation, protein expression and protein purification are carried out. The purified enzyme obtained has good thermal stability and also has the ability to efficiently synthesize carnosine; (2) The L-carnosine hydrolase mutant of the present invention, after protein expression and protein purification, the specific enzyme activity of the purified enzyme obtained is 1.23-2.75 times that of the wild-type thermophilic carnosine hydrolase. When catalyzing the synthesis of L-carnosine from β-alanine and L-histidine, the conversion rate of L-carnosine obtained can reach up to 46.7% at most. Description of the Drawings

[0011] Figure 1 It is the specific enzyme activity results of the wild-type TcPepD, N117E mutant, I169L mutant, and N117E / I169L double mutant in Example 4. Detailed Embodiments

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with specific related embodiments. However, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0013] Example 1 Construction of the Thermophilic Carnosine Hydrolase Expression Strain The wild-type of the thermophilic carnosine hydrolase is derived from Thermus thermophilus Thermobrachium celere The amino acid sequence is shown in SEQ ID NO.1 in the sequence listing, and the corresponding nucleotide sequence is shown in SEQ ID NO.2 in the sequence listing. The nucleotide sequence was fully synthesized and cloned between the restriction endonuclease sites BamHI and HindIII of the pET-28a vector to obtain the recombinant plasmid pET28a-TcPepD, which was further transformed into the expression host E.coli BL21(DE3), and positive clones were picked to obtain the recombinant expression transformant E.coliBL21(DE3) / pET-28a-TcPepD。

[0014] Example 2 Site-directed Mutagenesis of Thermophilic Carnosinase, Recombinant Expression Plasmid and Construction of Engineering Bacteria Some potential mutation sites for improving enzyme activity can be screened through molecular docking and substrate channel prediction. In this example, the amino acid sites M80, N117, N168, I169, E172, C181, and V459 were screened and mutated by the above methods. The sequences of the mutation primers are as follows: M80RF: As shown in SEQ ID NO.3 in the sequence listing; M80RR: As shown in SEQ ID NO.4 in the sequence listing; N117EF: As shown in SEQ ID NO.5 in the sequence listing; N117ER: As shown in SEQ ID NO.6 in the sequence listing; N168SF: As shown in SEQ ID NO.7 in the sequence listing; N168SR: As shown in SEQ ID NO.8 in the sequence listing; I169LF: As shown in SEQ ID NO.9 in the sequence listing; I169LR: As shown in SEQ ID NO.10 in the sequence listing; E172HF: As shown in SEQ ID NO.11 in the sequence listing; E172HR: As shown in SEQ ID NO.12 in the sequence listing; C181AF: As shown in SEQ ID NO.13 in the sequence listing; C181AR: As shown in SEQ ID NO.14 in the sequence listing; V459IF: As shown in SEQ ID NO.15 in the sequence listing; V459IR: As shown in SEQ ID NO.16 in the sequence listing; The above sequences were synthesized by NIOBIOTECH Co., Ltd. Using the single colony of BL21(DE3) / pET-28a-TcPepD as a template, amplification was carried out with the corresponding primers. The reaction system was: 2 μL of template; 25 μL of Primer STAR Max DNA polymerase; 20 μMol / L each of forward and reverse primers; add sterile water to a total volume of 50 μL.

[0015] The reaction conditions were: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 4 min, for a total of 30 cycles; after completion, final extension at 72°C for 10 min, and incubation at 4°C.

[0016] The amplified product was verified by nucleic acid electrophoresis and then purified using a PCR Purification Kit. The recovered fragment was transformed into BL21(DE3) competent cells, which were then spread on an LB plate (containing 50 μg / mL Kanamycin). Positive transformants were screened out, and recombinant plasmids were extracted separately and sent to Qingdao Weilai Biotechnology Co., Ltd. for sequencing verification. The sequencing results showed that the mutation was correct and no other random mutations occurred.

[0017] Example 3 Protein expression and protein purification of different mutant strains The different recombinant bacteria obtained in Example 1 and Example 2 were inoculated into 50 mL of LB liquid medium containing 50 μg / mL Kanamycin and cultured with shaking at 37 °C and 220 rpm for 8 h. Subsequently, the bacterial cells were transferred to 100 mL of LB liquid medium without Kanamycin and cultured at 37 °C and 220 rpm until OD600 = 0.8. IPTG with a final concentration of 0.2 mM was added, and induction was carried out at 18 °C and 220 rpm for 16 h. The cells were collected by centrifugation at 6000 rpm for 10 min.

[0018] The protein was purified using Ni-NTA nickel column affinity chromatography. The bacterial cells were resuspended in 50 mL of buffer (20 mM Tris-HCl; 250 mM NaCl; 10 mM imidazole; pH 8.0), and then disrupted using a pressure crusher. After centrifugation at 4 °C and 8000 rpm for 20 min, the supernatant was loaded onto a nickel column that had been equilibrated with Tris-HCl buffer. The adsorbed protein was eluted with a gradient of loading buffer containing different concentrations of imidazole (0 - 150 mM). The eluate of each elution peak was collected and identified by SDS-PAGE. The protein with the same size as the target size was centrifugally concentrated. The purified protein was used as the purified enzyme and stored at -80 °C with 25% glycerol at a final concentration.

[0019] Example 4 Activity detection of L-carnosine hydrolase enzyme synthesis reaction The synthetic activity was measured separately in a 200 μL Tris-HCl (50 mM, pH 8.0) reaction system, which contained 0.05 g / L of the purified enzyme obtained in Example 3, 2.0 M β-alanine, and 0.1 M L-histidine. The reaction was carried out at 70 °C for 10 min. 200 μL of 1 M HCl was added to 200 μL of the reaction solution to quench the reaction, and the enzyme activity was calculated by analyzing the concentration of the generated L-carnosine by HPLC external standard method.

[0020] The specific enzyme activities of the mutants obtained from the mutation sites M80, N168, E172, C181, and V459 were all lower than that of the wild-type TcPepD. The specific enzyme activity of the wild-type TcPepD was 1.58 U / mg, and the specific enzyme activities of the N117E mutant and the I169L mutant were 2.97 U / mg and 1.95 U / mg, respectively, which were 1.88 times and 1.23 times that of the wild-type TcPepD. The amino acid sequence of the N117E mutant was as shown in SEQ ID NO.17 in the sequence listing, and the nucleotide sequence was as shown in SEQ ID NO.18 in the sequence listing; the amino acid sequence of the I169L mutant was as shown in SEQ ID NO.19 in the sequence listing, and the nucleotide sequence was as shown in SEQ ID NO.20 in the sequence listing.

[0021] According to the method of Example 2, double mutations were performed on the amino acid sites N117E and I169L to obtain the N117E / I169L double mutant. The amino acid sequence of the N117E / I169L double mutant was as shown in SEQ ID NO.21 in the sequence listing, and the nucleotide sequence was as shown in SEQ ID NO.22 in the sequence listing.

[0022] After protein expression and protein purification of the N117E / I169L double mutant according to the method of Example 3, a purified enzyme was obtained, and then the specific enzyme activity was tested. The specific enzyme activity of the N117E / I169L double mutant was 4.35 U / mg, which was 2.75 times that of the wild-type. The specific specific enzyme activity results are shown in Figure 1 as shown.

[0023] For the synthesis reaction, one unit of enzyme was defined as the amount of enzyme that produced 1.0 μmol L-Car per minute under the above conditions.

[0024] Example 5 Catalytic Synthesis of L-Carnosine by Carnosine Hydrolyase The N117E / I169L double mutant with the highest specific enzyme activity in Example 4 was subjected to protein expression and protein purification according to the method of Example 3 to obtain a purified enzyme, and then L-carnosine was catalytically synthesized according to the following method: 6M β-alanine, 0.15M L-histidine, and 0.1 mM MnCl2 were added to Tris-HCl buffer solution (pH 8.5, 100 mM), and then 0.05 g / L of the purified enzyme was added. The total reaction volume was 0.5 mL. The reaction was carried out on a constant temperature metal bath at 70 °C and 1000 rpm for 10 h, and the reaction was terminated with 0.5 mL of 1M HCl to obtain 70 mM carnosine, and the yield based on histidine was 46.7%.

[0025] In summary, the wild-type of L-carnosine hydrolase is derived from Thermus thermophilus Thermobrachium celere, a mutant protein with mutations in the core amino acids related to enzyme catalytic activity is obtained after site-directed mutagenesis, which has significantly improved activity in catalyzing β-alanine and L-histidine. The L-carnosine hydrolase mutants include, but are not limited to, single-site mutations and multi-site mutations based on this patent.

Claims

1. A thermophilic L-carnosine hydrolase mutant, characterized in that, The thermophilic L-carnosine hydrolase mutant is one of the N117E mutant, the I169L mutant or the N117E / I169L double mutant; The amino acid sequence of the N117E mutant is shown as SEQ ID NO.17 in the sequence listing, the amino acid sequence of the I169L mutant is shown as SEQ ID NO.19 in the sequence listing, and the amino acid sequence of the N117E / I169L double mutant is shown as SEQ ID NO.21 in the sequence listing.

2. The thermophilic L-carnosine hydrolase mutant according to claim 1, characterized in that, The nucleotide sequence of the N117E mutant is shown as SEQ ID NO.18 in the sequence listing; the nucleotide sequence of the I169L mutant is shown as SEQ ID NO.20 in the sequence listing; the nucleotide sequence of the N117E / I169L double mutant is shown as SEQ ID NO.22 in the sequence listing.

3. Use of the thermophilic L-carnosine hydrolase mutant according to any one of claims 1-2 in the preparation of L-carnosine, characterized in that, After protein expression and protein purification of the thermophilic L-carnosine hydrolase mutant, a purified enzyme is obtained. Then, using β-alanine and L-histidine as substrates and a buffer solution with a pH of 6 - 10 as the reaction medium, the catalytic reaction is carried out using the purified enzyme at 50 - 80 °C to obtain L-carnosine.

4. Use of the thermophilic L-carnosine hydrolase mutant according to claim 3 in the preparation of L-carnosine, characterized in that, The concentration of the β-alanine is 0.2 - 6 M; The concentration of the L-histidine is 0.1 - 0.2 M; The concentration of the purified enzyme is 0.04 - 0.06 g / L; The rotation speed of the catalytic reaction is 1000 rpm, and the reaction time is 1 - 24 h.

5. Use of the thermophilic L-carnosine hydrolase mutant according to claim 3 in the preparation of L-carnosine, characterized in that, MnCl2 is also added in the catalytic reaction, and the concentration of MnCl2 is 1 - 100 mM.

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